DNA barcodes and cryptic species of skipperbutterflies in the genus Perichares in Areade Conservacion Guanacaste, Costa RicaJohn M. Burns*†, Daniel H. Janzen†‡, Mehrdad Hajibabaei§, Winnie Hallwachs‡, and Paul D. N. Hebert§

*Department of Entomology, National Museum of Natural History, Smithsonian Institution, P.O. Box 37012, MRC 127, Washington, DC 20013-7012;‡Department of Biology, University of Pennsylvania, Philadelphia, PA 19104; and §Biodiversity Institute of Ontario, Department of Integrative Biology,University of Guelph, Guelph, ON, Canada N1G 2W1

Contributed by Daniel H. Janzen, December 23, 2007 (sent for review November 16, 2007)

DNA barcodes can be used to identify cryptic species of skipperbutterflies previously detected by classic taxonomic methods andto provide first clues to the existence of yet other cryptic species.A striking case is the common geographically and ecologicallywidespread neotropical skipper butterfly Perichares philetes (Lep-idoptera, Hesperiidae), described in 1775, which barcoding splitsinto a complex of four species in Area de Conservacion Guanacaste(ACG) in northwestern Costa Rica. Three of the species are new,and all four are described. Caterpillars, pupae, and foodplants offerbetter distinguishing characters than do adults, whose differencesare mostly average, subtle, and blurred by intraspecific variation.The caterpillars of two species are generalist grass-eaters; of theother two, specialist palm-eaters, each of which feeds on differentgenera. But all of these cryptic species are more specialized in theirdiet than was the morphospecies that held them. The four ACGtaxa discovered to date belong to a panneotropical complex of atleast eight species. This complex likely includes still more species,whose exposure may require barcoding. Barcoding ACG hesperiidmorphospecies has increased their number by nearly 10%, anunexpectedly high figure for such relatively well known insects.

caterpillars � foodplants � genitalia � species diversity � taxonomy

Compared with most insects, skipper butterflies (Hesperi-idae) are taxonomically well known. Worldwide in occur-

rence, they are especially rich in neotropical species. Many ofthese are small, drab, and nondescript, but some are large,colorful, and showy enough to have been illustrated and formallynamed two or more centuries ago. Since then, a specimen thatsuperficially seemed to fit a named species has often beenassigned to it. As a consequence, the geographic distributionascribed to a named species has usually grown over time, andrepeated application of the specific epithet has tended tobroaden the concept of that species. One outcome is a common,widespread, somewhat variable, but readily recognizable taxonthat has become too familiar to generate further taxonomicinterest. Such ‘‘weed species’’ warrant closer examination.

A prime example is Astraptes fulgerator, a large, common,flashy skipper described in 1775 and thought by mid-20thcentury to be panneotropical in distribution (1). When reared ingreat numbers from wild-caught dicot-eating caterpillars in Areade Conservacion Guanacaste (ACG) of northwestern CostaRica, this skipper seemed much too polyphagous for a singlespecies. Although initial morphological examination was incon-clusive, detailed comparison of large samples of adults, groupedfirst by what they had eaten as larvae and then by sex, revealedsix or seven species differing in extremely subtle traits (e.g.,shades of blue and yellow, wing shape, and size) that covariedwith some ecological preferences (dry vs. rain vs. cloud forest)and different color patterns of supposedly polymorphic cater-pillars. Subsequent cytochrome c oxidase (COI) DNA barcoding(2) of 466 reared adults yielded clusters that covaried with thosealready detected, plus three new clusters, two derived from small

adult samples and one mostly from wild-caught pupae [larvalfoodplant(s) unknown at that time], for a total of 10 crypticspecies that range from parapatric to sympatric (3).

Author contributions: J.M.B., D.H.J., M.H., W.H., and P.D.N.H. designed research; J.M.B.,D.H.J., M.H., W.H., and P.D.N.H. performed research; J.M.B., D.H.J., M.H., W.H., and P.D.N.H.contributed new reagents/analytic tools; J.M.B., D.H.J., M.H., W.H., and P.D.N.H. analyzeddata; and J.M.B., D.H.J., M.H., W.H., and P.D.N.H. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

Data deposition: The sequences reported in this paper have been deposited in the GenBankdatabase (for accession nos., see SI Table 2).

†To whom correspondence may be addressed. E-mail: burnsj@si.edu or djanzen@sas.upenn.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0712181105/DC1.

© 2008 by The National Academy of Sciences of the USA

Fig. 1. NJ tree based on Kimura two-parameter distances for COI DNAbarcodes of the six species of Perichares reared in ACG; upper four species(three new) belong to the P. philetes species complex. Numbers indicate howmany individuals with each haplotype; colored bars indicate ecosystem occur-rence and larval foodplants; red dots indicate the haplotypes of holotypes ofthe three new species.

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1 4 7 10

2 5 8 11

3 6 9 12

14

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13

Fig. 2. Last-instar caterpillar (in anterior and dorsal view of same individual) and pupa [in dorsal, or dorsolateral, view of different individual (except for P.prestoeaphaga)] of the six species of Perichares reared in ACG. Voucher code of each individual in parentheses. (1–3) P. adela (03-SRNP-6347, 06-SRNP-30496).(4–6) P. poaceaphaga (06-SRNP-30259, 06-SRNP-30375). (7–9) P. geonomaphaga (05-SRNP-4067, 00-SRNP-11394). (10–12) P. prestoeaphaga (00-SRNP-11749).(13) P. deceptus (03-SRNP-4930). (14–17) P. lotus [04-SRNP-11636 (including lateral view of head), 04-SRNP-13892].

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In another analysis of reared ACG skippers, four new and verysimilar species (limited to rain and cloud forest) in the suppos-edly monotypic genus Venada were characterized by moderatedifferences in adult facies and male and female genitalia and bymajor differences in the color pattern of last-instar caterpillars.Subsequent barcoding conspicuously separated these species (4).

However, barcodes seemed not to separate closely relatedspecies within each of three pairs of skippers in the generaPolyctor, Cobalus, and Neoxeniades (5), even though notabledifferences in ecology (one species in ACG dry forest, the otherin adjacent rain forest) and in morphology (facies, genitalia)show that the species are undeniably distinct. This apparent lackof resolution came from the use of DNA barcodes too short toinclude diagnostic sites. Full-length barcodes (�650 base pairs)reliably distinguished the species in each pair of skippers, withthe differences involving just one to three nucleotides. Although,in some other species, such minor barcode variation is individual,this study clearly shows that setting some arbitrary level ofdifferentiation below which individuals are considered conspe-cific is untenable (6).

In each of the preceding studies, barcodes were an addedcharacter that reinforced and expanded prior conclusionsreached by traditional taxonomic means. But barcodes can alsoprovide the very first clue to the existence of unsuspectedspecies.

In its beginning stages, a program of systematically barcodingevery ACG macrolepidopteran species (5, 7) included a fewspecimens of the skipper Perichares philetes. This is a large,common, distinctive, monocot-eating ‘‘weed species’’ that cur-rent taxonomy treats as polytypic and panneotropical. Unex-pectedly, the first two P. philetes specimens to be barcodedshowed a deep divergence within their conspecific cluster in aneighbor-joining (NJ) tree. This stimulated progressively moresearch for P. philetes caterpillars, more barcoding of P. philetesadults, and scrutiny of all life stages. Subsequent NJ trees oflarger and larger samples (totaling 255 reared wild-caughtindividuals through mid-2007) revealed four indisputable clus-ters [Fig. 1 and supporting information (SI) Appendices 1 and 2].

With the circumstances reversed, the obvious taxonomic ques-tion is: What other kinds of evidence justify formal recognition ofthe cryptic species of Perichares indicated by DNA barcodes? Astrong supportive character is larval foodplants: although all fourspecies feed on monocots, two eat grasses, and two eat palms. Thetwo grass-eaters use a diversity of grasses (including a few intro-duced species), overlap in their foodplant and microhabitat choices,and occur in both rain and dry forest; one also eats sedges.However, each of the two palm-eaters specializes on one or twospecies in a different genus of sympatric palms (even though manyspecies of palms are available) and occurs only in rain forest (despitethe presence of three species of indigenous palms in adjacent ACG

Fig. 3. Males (columns one and three) and females (columns two and four) of four cryptic species of Perichares in dorsal (left) and ventral (right) view. Vouchercode of each specimen in parentheses or brackets. (1–4) P. adela (06-SRNP-45166, 06-SRNP-40498). (5–8) P. poaceaphaga [06-SRNP-1189 (holotype), 03-SRNP-23215]. (9–12) P. geonomaphaga [04-SRNP-2706 (holotype), 04-SRNP-30964]. (13–16) P. prestoeaphaga [01-SRNP-22227 (holotype), 06-SRNP-31113].

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dry forest) (see http//:janzen.sas.upenn.edu and SI Appendix 1 andSI Table 2 for all specimen collateral information).

Differences in the color patterns of last-instar caterpillars andpupae further support species status (Fig. 2 1–12). In both stages,a pair of dorsal, longitudinal, close-set, yellow stripes runs thelength of a green body. The stripes of caterpillars vary in bothdefinition and width (stripes of grass-eaters are fuzzier and widerthan are those of palm-eaters), whereas the dorsal stripes ofpupae vary in their continuity and, to a lesser extent than incaterpillars, in width. Intensity of pigmentation of the caterpillarhead also varies interspecifically, from very pale cream (overpale green), through medium tan, to rusty brown.

Adults, on the contrary, are nearly indistinguishable in facies,even when sorted by their barcodes and sex (Fig. 3). Large sortedsamples of reared specimens (with notably unworn wings)appear, en masse, to differ, but mostly in ways so subtle andvariable that definitive characterization is elusive. The speciesdiffer slightly in average size, but the degree of overlap is high.Morphological identification of an isolated individual, especiallyif wild-caught (caterpillar foodplant unknown) and somewhatworn, is unreliable.

Adults are sexually dimorphic with respect to the positions ofyellow spots on the forewing, and males have a sizable, curved,gray, secondary sex character (Fig. 3 1, 5, 9, and 13) between theouter spots and the inner one. Many species of skippers aresexually dimorphic in wing shape, with the wings of malesappreciably narrower and more pointed (and the hindwingssometimes longer) than those of females. The two palm-eatingspecies unmistakably express this wing-shape dimorphism (oneof them more perceptibly than the other), whereas the grass-eaters do not (males more closely resemble females). Exceed-ingly subtle sexual dimorphism relates to the quality of yellow inthe forewing spots: usually, although not invariably, the yellowappears to be slightly darker in males but paler in females, exceptfor one of the grass-eaters, in which the yellow usually appearspale in both sexes.

Within each sex, forewing spots vary considerably in size andshape, but the variation is individual. What matters taxonomi-cally is the expression on the dorsal (although not the ventral)surface of the forewing of the lowest spot, which is relativelysmall and located slightly above the posterior edge of the wing.On average, this spot is better expressed in the grass- than in thepalm-eaters, in one of which the spot hardly ever appears, andthen only in males. On average, checkering of wing fringes ismore pronounced in grass- than in palm-eaters.

Genitalia (which often distinguish related species) seem, likefacies, too similar and individually variable to be dependablydiagnostic for these four cryptic species. Compare minor, vari-able, genitalic differences between individuals of the four specieswith the obvious differences that characterize the other twospecies of Perichares in ACG (see Fig. 1 and SI Figs. 4 and 5).

One of the grass-eaters is by far the most common andpolyphagous of the four similar species indicated by barcodes. Itis not surprising that this grass-eater and occasional sedge-eatercorresponds morphologically with what is currently classified asan extremely widespread, continental, panneotropical subspe-cies, P. philetes adela [ACG material matches the type of thistaxon (described in 1867 from Rio de Janeiro, Brazil) in theNatural History Museum, London]. The other three species areundescribed. The specific name (a noun in apposition) given toeach conveys dietary preference (e.g., the more scarce grass-eater is Perichares poaceaphaga). All four species are succinctlyand comparatively described in Table 1. Although the twograss-eaters are ecologically and morphologically the most sim-ilar species, they are farthest apart in the NJ tree (compare Fig.1 and Table 1; see also SI Appendices 1 and 2).

Apropos of Table 1, a cautionary note: The samples of adultsare large enough to show individual variation, and it is clearly

pervasive. Although reflecting real differences, the averages ofwing length in the species descriptions are low, because rearedadults average smaller than wild ones. Each caterpillar receivesthe species of plant on which it was found, but new foliage isoffered only at 3- to 4-day intervals, and its quality is thereforeinferior. The degree to which a reared adult is stunted dependsat least in part on the instar of the caterpillar when it wascollected and hence on how long it has been fed in captivity.Wild-caught caterpillars in our samples far outnumber wild-caught pupae, which presumably yield adults of natural size.Perichares follows the general rule in skippers that females areon average larger than males (for detailed documentation inunrelated hesperiid genera, see refs. 8 and 9).

Two other species of Perichares, both grass-eaters, have beenreared (rarely) in ACG. Adults of these species differ conspic-uously from each other and from the cryptic four (Fig. 1) (SIAppendix 2, SI Figs. 4 and 5). Although the last-instar caterpillarof Perichares deceptus has a pair of dorsal yellow stripes, theyembrace a narrowly black-bordered blue central stripe (Fig. 213). When Perichares lotus pupates, it shows its stripes (Fig. 2 17),but the caterpillar lacks them (Fig. 2 14), and the lower side ofits head has a large, elliptical, black spot (Fig. 2 16), which doesnot appear in the other species.

DiscussionTo a great extent, current skipper classification (10) still reflectssweeping revisionary work of the mid-20th century (1), in whichrelated species were often reduced to subspecific rank or orig-inally described as subspecies, without adequate justification (11,12). Thus, P. philetes became a polytypic species of four subspe-cies: two long-known and wide-ranging in the neotropics and twonewly described and limited in distribution (essentially to south-ern Brazil plus Paraguay and to Peru) (1). Judging from facies,the latter are distinct species, Perichares aurina and Perichareslimana, as is a supposed synonym of Perichares adela calledPerichares marmorata (originally described from Venezuela).There is some sympatry among taxa. All of the above, togetherwith the three new species from ACG, now constitute the P.philetes species complex.

P. philetes, originally described from Jamaica in 1775, is aGreater Antillean grass-eater. Like the grass-eaters in ACG,its mature caterpillar has a pair of fuzzy, wide, close-set, yellowstripes, and its pupa has ‘‘two longitudinal and parallel lightyellow lines or stripes travers[ing] the dorsal surface’’ (13).Caterpillars have repeatedly been found feeding on sugar cane(Saccharum), an introduced grass, originally from Asia, inCuba (14), Jamaica (13), Hispaniola (15), and Puerto Rico (16,17). This seeming monophagy is an artifact stemming from theeconomic importance of the food/plant. In Jamaica, caterpil-lars occasionally were found on Panicum maximum and Zea(13) and, in Puerto Rico, on two bamboos, Bambusa vulgarisand Cephalostachyum pergracile (18). When hurricane Hugodevastated forests in northeastern Puerto Rico in September1989, P. philetes caterpillars were found the next spring in largenumbers on the sedge Scleria pterota, which f lourished indisturbed areas; ‘‘some plants were attacked by eight or morelarvae’’ (19). Species of Saccharum, Panicum, and Scleriaare among the many foodplants of Perichares adela in ACG(Table 1).

In central Brazil, ‘‘the larva [of P. philetes], though sometimesfound on [a single species in each of two genera (Desmoncus andHyospathe) of palms] appears to favour the common sugar canein the neighbourhood of Para’’ (20). There are additional recordsof P. philetes on both palm and grass in Trinidad and Guyana and,again, on sugar cane in Trinidad, Venezuela, Guyana, andArgentina (18). At the very least, two species are involved. Thename P. philetes does not refer to the palm-eaters, and whetherit can be applied to the grass-eaters is questionable. P. philetes

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Table 1. Perichares philetes species complex reared from wild-caught caterpillars in ACG, northwestern Costa Rica, including numbersof caterpillars found on each species of foodplant

CharactersP. adela

(Hewitson)P. poaceaphaga

Burns n. sp.P. geonomaphaga

Burns n. sp.P. prestoeaphaga

Burns n. sp.

Larval foodplantsCyperaceae

Scleria latifolia 1Scleria mitis 1Scleria sp. 1

PoaceaeArundinella deppeana 6 2Brachiaria sp. 2Lasiacis procerrima 1Lasiacis sorghoidea 1Megathyrsus maximus* 18 5Oryza latifolia 2Panicum vulgaris 1Paspalum botteri 3Paspalum nutans 1Paspalum virgatum 62 3Paspalum sp. 3Rottboellia cochinchinensis* 5 1Saccharum spontaneum* 2Setaria paniculifera 2 27Urochloa arrecta 4 1Undetermined spp. 10 2

ArecaceaeAstrocaryum alatum 2Chamaedorea deckeriana 1Geonoma congesta 17Geonoma interrupta 49Prestoea decurrens 61

Ecosystem Dry and rainforest

Dry and rainforest

Rain forest Rain forest

Caterpillar, last instarHead color Very pale tan

(on pale green)(Fig. 2 1)

Pale tan(Fig. 2 4)

Rusty brown(Fig. 2 7)

Tan (Fig. 2 10)

Body, yellow stripes Very wide,fuzzy (Fig. 2 2)

Wide, fuzzy(Fig. 2 5)

Narrow, quite sharp(Fig. 2 8)

Narrow, sharp(Fig. 2 11)

PupaBody, yellow stripes Continuous,

very narrow(Fig. 2 3)

Continuous,narrow

(Fig. 2 6)

Continuous,narrow

(Fig. 2 9)

Dashed, wide(Fig. 2 12)

AdultForewing length, mm � 21.46, 0.910 � 22.40, 1.120 � 23.47, 0.862 � 23.43, 1.813

19.7–23.3, 55 19.6–23.9, 24 22.2–25.0, 13 20.1–25.3, 9Mean, SDRange, n � 23.31, 1.056 � 23.99, 0.955 � 25.54, 1.364 � 25.95, 1.895

20.2–26.0, 54 21.8–25.6, 19 22.5–27.9, 21 21.3–29.0, 11

Hindwing shape � Closer to �

(Fig. 3 1, 3)Closer to �

(Fig. 3 5, 7)Elongate, quite narrow

(Fig. 3 9, 11)Elongate, narrow

(Fig. 3 13, 15)Forewing yellow spots � darker � lighter � and � light � darker, � lighter � darker, � lighterForewing lowest spot,

dorsal expression ofUsually sizable

(Fig. 3 1, 2)Sizable

(Fig. 3 5, 6)Usually smaller(Fig. 3 9, 10);absent in 5 �

Absent in � and inmost � (Fig. 3 13, 14);

a pinpoint in 3 �

Wing fringe checkering Strong (Fig. 3 1–4) Strong (Fig. 3 5–8) Weak (Fig. 3 9–12) Weak (Fig. 3 13–16)

Holotype � 06-SRNP-1189 04-SRNP-2706 01-SRNP-22227

Paratypes 22 �, 17 � 11 �, 18 � 5 �, 8 �

See SI Table 2 for specimen detail.*Introduced.

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was reported from northwestern Argentina (21), and subspeciesP. philetes philetes, from the Greater Antilles, on the one hand,and Trinidad, Tobago, the Guianas, central Brazil (Pernambuco,Para, Amazonas), and Iquitos, Peru, on the other (1). But thesouthern continental populations are widely separated from thenorthern insular populations and may well be specifically dis-tinct.

Grass-eaters of the P. philetes complex appear to be oppor-tunistic generalists, whereas palm-eaters are narrow specialists.However, these specialists, unlike those in the A. fulgeratorcomplex (3), use a single plant family (Arecaceae) instead of adiversity of unrelated plant families. That many tropical speciesthought to be dietary generalists actually include or comprisesets of dietary specialists is becoming increasingly evident, inpart because of barcoding efforts (e.g., ref. 22). In the course ofbarcoding 422 morphospecies of ACG skippers, �40 (�9.5%)seem to be two or more biological species. P. philetes is aspectacular case, second only to that of A. fulgerator (3).

Revelation and analysis of the P. philetes species complex areincomplete, because barcodes have been obtained inside but notoutside of ACG. The massive and intensive endeavor of the ACGbioinventory cannot do both. Nevertheless, a major effort ismade to sample more ACG specimens whenever incongruitiesappear in a NJ tree. The four species of the P. philetes complexin ACG are so irrefutably real that they must occur beyond thissmall area of 125,000 terrestrial ha, and some of their characters(including barcodes) may vary geographically. For now, attemptsto place these species in a broader taxonomic and geographic

context can only rely on fragmentary nonmolecular evidence. Inits entirety, the complex probably includes additional crypticspecies. All things considered, the bioinventory of ACG isopening a can of neotropical worms, or caterpillars, and cata-lyzing taxonomic metamorphoses.

Materials and MethodsFor specifics relating to specimen collection and molecular analyses, see ref. 5.

Specimen images, collection details, and sequence records are also avail-able in the project file ‘‘Perichares of the ACG’’ on the Barcode of Life DataSystem web site (www.barcodinglife.org) (see ref. 5). All barcoded specimens(including holotypes) have been deposited in the National Museum of NaturalHistory, Smithsonian Institution, and may be recovered by their individualvoucher codes (SI Table 2). Barcodes of holotypes and of a representativespecimen of P. adela are given in SI Table 3).

ACKNOWLEDGMENTS. We thank Sarah Burns for helping in many and variousways; Young Sohn for drawing genitalia figures; Karie Darrow for arrangingthem in plates and fitting Table 1 on a single page; Lee-Ann Hayek forcalculating statistics; Donald Harvey for dissecting genitalia and locatingliterature; Campbell Smith for photographing holotypes in the Natural HistoryMuseum, London; Tanya Dapkey for delegging and documenting specimens;Rebecca Cowling for assisting with molecular analysis; and ACG parataxono-mists for finding and rearing the caterpillars. This work was supported by theNational Museum of Natural History Small Grants Program (J.M.B.); by U.S.National Science Foundation Grants BSR 9024770 and DEB 9306296, 9400829,9705072, 0072730, and 0515699; by grants from the Guanacaste Dry ForestConservation Fund and ACG (D.H.J.); and by the Gordon and Betty MooreFoundation, the Natural Sciences and Engineering Research Council of Can-ada, Genome Canada through the Ontario Genomics Institute, and the Can-ada Research Chairs program (P.D.N.H.).

1. Evans WH (1955) A Catalogue of the American Hesperiidae Indicating the Classifica-tion and Nomenclature Adopted in the British Museum (Natural History), Part IV,Hesperiinae and Megathyminae (British Museum, London), pp 1–499, pls 54–88.

2. Hebert PDN, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications throughDNA barcodes. Proc R Soc London Ser B 270:313–321.

3. Hebert PDN, Penton EH, Burns JM, Janzen DH, Hallwachs W (2004) Ten species in one:DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptesfulgerator. Proc Natl Acad Sci USA 101:14812–14817.

4. Burns JM, Janzen DH (2005) Pan-neotropical genus Venada (Hesperiidae: Pyrginae) isnot monotypic: four new species occur on one volcano in the Area de ConservacionGuanacaste, Costa Rica. J Lepid Soc 59:19–34.

5. Hajibabaei M, Janzen DH, Burns JM, Hallwachs W, Hebert PDN (2006) DNA barcodesdistinguish species of tropical Lepidoptera. Proc Natl Acad Sci USA 103:968–971.

6. Burns JM, Janzen DH, Hajibabaei M, Hallwachs W, Hebert PDN (2007) DNA barcodes ofclosely related (but morphologically and ecologically distinct) species of skipper but-terflies (Hesperiidae) can differ by only one to three nucleotides. J Lepid Soc 61:138–153.

7. Janzen DH, Hajibabaei M, Burns JM, Hallwachs W, Remigio E, Hebert PDN (2005)Wedding biodiversity inventory of a large and complex Lepidoptera fauna with DNAbarcoding. Philos Trans R Soc London Ser B 360:1835–1845.

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15. Wolcott GN (1922) The insects of sugar cane in Santo Domingo. J Dept Agric LaborPorto Rico 6:32–37.

16. Jones TH, Wolcott GN (1922) The caterpillars which eat the leaves of sugar cane in PortoRico. J Dept Agric Labor Porto Rico 6:38–50.

17. Wolcott GN (1951) The insects of Puerto Rico. J Agric Univ Puerto Rico 32:417–748.18. Cock MJW (2005) The skipper butterflies (Hesperiidae) of Trinidad. Part 13, Hesperi-

inae, genera group K. Living World, J Trinidad Tobago Field Nat Club 2005:23–47.19. Torres JA (1992) Lepidoptera outbreaks in response to successional changes after the

passage of Hurricane Hugo in Puerto Rico. J Trop Ecol 8:285–298.20. Moss AM (1949) Biological notes on some ‘‘Hesperiidae’’ of Para and the Amazon (Lep.

Rhop.). Acta Zool Lilloana 7:27–79, pls I–V.21. Hayward KJ (1950) Insecta, Lepidoptera (Rhopalocera), familia Hesperiidarum, sub-

familia Hesperiinarum, in Genera et Species Animalium Argentinorum, ed Descole HR(Guillermo Kraft, Buenos Aires), 2:1–388, pls I-XXVI.

22. Smith MA, Wood DM, Janzen DH, Hallwachs W, Hebert PDN (2007) DNA barcodesaffirm that 16 species of apparently generalists tropical parasitoid flies (Diptera,Tachinidae) are not all generalists. Proc Natl Acad Sci USA 104:4967–4972.

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